Methods of treatment of xerophthalmia with self-preserving ocular formulations of norketotifen

ABSTRACT

Self-preserving ophthalmic formulations containing norketotifen and methods of making the same and the use thereof in patients suffering from xerophthalmia are disclosed. The methods also comprise administering to the eyes of a mammal in need thereof, self-preserving topical ophthalmic formulations containing norketotifen, free from any added preservative.

This application claims priority of Provisional Application Ser. No.61/199,883, filed Nov. 21, 2008, the disclosure of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compositions for administration of thecompound RS-norketotifen, hereinafter called norketotifen. This compoundis useful for treating a variety of ocular disorders, includingxerophthalmia and various forms of conjunctivitis and keratitis (U.S.Pat. No. 6,207,684).

Chemically, norketotifen is4-(4-piperidyliden)-9-oxo-9,10-dihydro-4H-benzo[4,5]cyclohepta[1,2-b]thiophene,which is a mixture of R-norketotifen and S-norketotifen.

A large number of excipients have now been tested for compatibility withnorketotifen and certain excipients have now been found to be compatiblewith norketotifen. Examples of compatible and incompatible excipients,such as for example members of the classes Antioxidants, Buffers,Chelating agents, Emollients, Gelling agents, Humectants, Mucoadhesiveagents, Preservatives, Solvents, Stabilizers, Surfactants, Tonicitymodifying agents and Viscosity modifying agents, have been identifiedand are described herein. Numerous compositions using said excipientshave been prepared and tested.

The terms “composition” and “formulation” are used as synonyms herein.The terms “about” and “approximately” are used as synonyms herein. Theterms “disorder” and “disease” are used as synonyms herein. The terms“carrier” and “solvent” are used as synonyms herein. The terms“norketotifen” and “norketotifen HF” as used herein refer to thehydrogen fumarate salt of said compound. The free base of norketotifenis herein called “norketotifen free base” or “norketotifen FB”. As usedherein, “about” means within the pharmaceutically acceptable limitsfound in the United States Pharmacopeia (USP-NF 21), 2003, or availableat www.usp.org for amount of pharmaceutical ingredients.

BACKGROUND OF THE INVENTION

Embodiments disclosed herein relate to compositions of norketotifen thatare intended for the treatment of ocular disorders, such asxerophthalmic disorders, allergic disorders and inflammatory disordersas well as combinations thereof, and methods of treating oculardisorders by administration of such compositions. The term“xerophthalmia” as used herein refers to conditions that are also calledxerophthalmic disorders, keratoconjunctivitis sicca, keratitis sicca,sicca syndrome, dry eye syndrome, or dry eyes. The present inventionalso relates to formulations of norketotifen that are intended for thetreatment of allergic (atopic) ocular diseases, such as for exampleallergic conjunctivitis and various other types of ocular inflammatorydiseases, such as for example keratitis, keratoconjunctivitis,non-allergic conjunctivitis and various forms of blepharitis(inflammation of the eyelids).

Norketotifen is an active metabolite of ketotifen (Zaditor®, Novartis),which is used as an ocular medication for allergic disorders.Norketotifen has antihistaminic activity (Kennedy G R, 1982) that isabout half of that of ketotifen when tested in vivo (Example 12.)Norketotifen is about ten times more active than ketotifen asanti-inflammatory compound (Example 13.)

Ketotifen is available commercially, for example from Sigma-Aldrich(Internet: Sigma-Aldrich.com/order). Norketotifen can be made bydemethylation of ketotifen according to the method described byWaldvogel et al. 1976, which publication is hereby incorporated byreference. However, this use of norketotifen is limited by its inherentirritability, as well as the irritability of certain excipients thatwere previously thought necessary to formulate an effective medicament,taking into account various issues including the limited solubility ofthe active ingredient norketotifen.

As described in U.S. Pat. No. 6,207,684, racemic norketotifen is usefulfor the treatment of various ocular conditions, such as for exampleconjunctivitis and keratitis. Further descriptions concerningS-norketotifen and R-norketotifen were presented in U.S. Pat. Nos.7,226,934 and 7,557,128, respectively. Said patents are herebyincorporated by reference. It has now been found that selectedformulations of norketotifen are well tolerated by mammalian oculartissues and have the surprising effect of decreasing or eliminating theocular irritating activity of norketotifen when applied to the eye. Saidselected formulations (herein called “preferred formulations”) ofnorketotifen are suitable for daily topical administrations. Ocularadministration of ophthalmic medication is the preferred route ofadministration.

Due to its physicochemical properties and its pharmacological effects,such as low anti-muscarinic activity, norketotifen is well suited forocular use.

To our knowledge, methods of administering norketotifen in a preferredformulation topically to the eye or into the conjunctival sac ofpatients suffering from eye disorders have not previously beendescribed. The therapeutic use of selected ocular formulationscontaining norketotifen has not been described, as formulations for theocular administration of norketotifen have previously not beendeveloped. Ocular formulations of ketotifen have been described (U.S.Pat. No. 6,455,547) but are of no relevance for norketotifen since thephysicochemical properties for norketotifen HF are vastly different fromthose of ketotifen—as an example, the water solubility for ketotifen isabout 12.5 mg/ml and the water solubility of norketotifen is about 2.16mg/ml.

Although hundreds of ophthalmic excipients exist, it is far from obviouswhat excipients may be compatible with norketotifen and whatcombinations of excipients and what concentrations thereof should beused to obtain optimal ophthalmic compositions that may optimize thatocular therapeutic activities and decrease the ocular side effects ofnorketotifen. This may be due to the fact that norketotifen hasphysicochemical, chemical and pharmacological properties and sideeffects that are unique to this specific molecule and therefore, newcompositions have to be developed for norketotifen. It is a well-knownfact that for example all the different ocular steroids need differentophthalmic formulations (compositions). Each composition is depending onthe physicochemical and the pharmacological properties of the activemolecule(s) and the therapeutic effects sought. In the case ofnorketotifen, our research has now arrived at compositions that areacceptable to the patients, therapeutically efficacious, allowing foronce-daily ocular administration or for repeated daily ocularadministrations to mammals in need thereof, while decreasing or eveninhibiting certain ocular side effects of norketotifen. Selected ocularcompositions have now been found, which offer pharmaceuticalcompatibility between the active ingredient and the excipients, optimizethe therapeutic activities of norketotifen and decrease the ocular sideeffects thereof.

The preferred formulations of the present invention, as described hereinare chemically stable and commercially feasible to manufacture.

It is an objective of the present invention to provide ocularcompositions that deliver therapeutically effective concentrations ofnorketotifen to the eye and the conjunctival tissues, which allow foronce-daily ocular administration or for repeated ocular administrationsfrom two to five times daily to a mammal in need thereof, while notcausing ocular side effects, such as burning, redness or irritation, andwhile at the same time being stable upon storage. The terms“therapeutically effective (dose)” and “therapeutically efficacious(dose)” refer to a dose that yields therapeutic benefit to patients,which in the present case refers to therapeutic benefit to patientssuffering from allergic and/or inflammatory condition(s).

The present invention may be useful for patients in need of medicationfor ocular inflammatory and ocular allergic disorders and for patientssuffering from xerophthalmic disorders. The term “patients” in thisdocument refers to mammals, such as for example humans, dogs and cats.Preferred are human patients.

Patients suffering from ocular allergic and/or inflammatory disordersinclude individuals being diagnosed as suffering, for example, fromvarious types of conjunctivitis, as for example allergic conjunctivitis,seasonal allergic conjunctivitis, chronic allergic conjunctivitis,atopic keratoconjunctivitis or vernal conjunctivitis. As used herein,the term “conjunctivitis” refers to all forms of the disease. Thesepatients often have symptoms, such as for example hyperemia of thebulbar conjunctiva, discharge, limbal erythema/swelling orerythema/swelling of the eyelids, itching, decreased lacrimation,photophobia, discomfort, foreign body sensation and/or xerophthalmia.

Keratitis is a condition in which the eye's cornea becomes inflamed.Various forms of keratitis exist, such as for example superficialkeratitis and deep keratitis. As used herein, the term “keratitis”refers to all forms of the disease.

The terms “dry eye disease” and “xerophthalmia” are used synonymouslyherein and refer to various disorders that are well known to thoseskilled in the art of ophthalmology. Examples of xerophthalmic disordersare keratoconjunctivitis sicca, age-related dry eye, Stevens-Johnsonsyndrome, Sjögren's syndrome, ocular cicatrical pemphigoid, blepharitis,corneal injury, infections, Riley-Day syndrome, congenital alacrima,nutritional disorders or deficiencies (including vitamin A deficiency),autoimmune and other immunodeficient disorder and side effects ofmedications such as for example anticholinergic drugs used foroveractive bladder syndrome and urinary urge incontinence. As usedherein, the term “dry eye disease” or “xerophthalmia” refer to all formsof the disease.

All compositions intended for use in the eye are required to be sterile,endotoxin-free and foreign particulate free. The term “foreignparticulate free” indicates the absence of any particulate matter, butexcludes drug particles, controlled release microparticulates and thelike. Conventional methods for the manufacture of sterile compositionsinclude sterilization by moist heat (autoclaving), sterilization by dryheat, ethylene oxide sterilization (gaseous sterilization), exposure toultraviolet rays or to gamma irradiation or sterilization by asepticprocessing or filtration through sterilizing grade filters.

Norketotifen formulations for ocular administration that are describedherein can be readily processed by standard manufacturing processes,which are well known to those skilled in the art.

SUMMARY OF THE INVENTION

The physicochemical properties of norketotifen have now beeninvestigated, with particular focus on the solubility and stabilityproperties of the compound. The compatibility of norketotifen with alarge number of ocular excipients has been studied. The optimalconcentration range, acidity range and tonicity range for ocularformulations of norketotifen have been determined from the presentstudies. It was determined that the concentration of norketotifen in thecompositions of the invention should be between 0.001 percent and 0.3percent, preferably between 0.01 percent and 0.25 percent. Formulationscontaining one or more solubilizing excipient may contain norketotifenin concentrations from 0.001 percent to 5.0 percent, more preferred from0.01 percent to 1.9 percent. The acidity of the final ocular compositionshould be between pH 4.0 to pH 6.5, preferably between pH 5.2 to pH 6.2.The tonicity of final aqueous ocular solutions should be from 150 mOsmto 450 mOsm and preferably from 230 mOsm to 330 mOsm.

Therapeutically effective ophthalmic compositions of norketotifen,containing excipients that are compatible with this drug have now beendeveloped and formulations, such as ocular solutions, ocular hydrophilicointments and gels, ocular hydrophobic ointments, ocular emulsions andocular liposome formulations, all containing norketotifen, have now beendeveloped and are described herein. It was an objective to developformulations of norketotifen that allow for once-daily ocularadministration and for repeated ocular administrations from two to fivetimes daily to patients in need thereof.

“Therapeutically effective” ocular solution formulations of norketotifenrefer to concentration of norketotifen from 0.001 percent to 0.30percent; more preferred are concentrations from 0.01 percent to 0.25percent by weight. “Therapeutically effective” formulations with one ormore solubilizing excipient refer to norketotifen concentrations from0.001 percent to 5.0 percent by weight, more preferred from 0.01 percentto 1.0 percent by weight.

An eyedropper device is usually used for the administration ofophthalmic solutions to the eye. The volume of each drop depends on theconstruction of the device, the technique used to produce the drop andthe viscosity of the solution being administered. Commercial eyedroppersusually deliver drops with a volume of 50 μL. Thus one drop ofnorketotifen 0.1 percent equals an amount of 50 μg norketotifen.

A squeezable tube with a small tip is usually used for theadministration of gels or ointments to the eye. The amount administereddepends on the technique used and the design of the tube. The amount ofthe gel or ointment dosed is usually from about 20 mg to about 50 mg foreach application.

During biological testing it was surprisingly found that the preferredformulations described herein completely protected the ocular tissuesfrom irritation by norketotifen, even if the highest possibleconcentrations of norketotifen were used.

Microbiological testing, using standard preservative challenge tests,surprisingly demonstrated self-preservation qualities of the preferrednorketotifen formulations. Thus, said formulations can be used withoutadded preservatives.

DETAILED DESCRIPTION OF THE INVENTION

The term norketotifen as used herein relates to the basic compounditself as well to any pharmaceutically acceptable salt thereof.Preferred pharmaceutically acceptable salts of norketotifen are forexample a hydrogen fumarate, a hydrochloride, a hydrobromide, a hydrogenmaleate, or a hydrogen sulfate. More preferred salts of norketotifen arethe hydrochloride salts and hydrogen fumarate salt. Most preferred isthe hydrogen fumarate salt. The term “pharmaceutically acceptable salt”and the like refer to salts prepared from pharmaceutically acceptableacids, such as for example fumaric, hydrobromic, hydrochloric, maleicand sulphuric acids.

The term “therapeutically effective ocular formulations of norketotifen”refers to solutions containing norketotifen in concentrations from about0.001 percent to about 0.30 percent; more preferred are concentrationsfrom 0.01 percent to 0.250 percent. Formulations containing one or moreexcipient with solubilizing activity may contain higher concentrationsof norketotifen than indicated above. Thus, for example gels, ointmentsand solutions containing solubilizing excipients may containnorketotifen in concentrations in excess of 5.0 percent. Therefore,“therapeutically effective” ocular formulations containing one or moresolubilizing excipient contain norketotifen in concentrations from about0.001 percent to about 5.0 percent, more preferred are concentrationsfrom about 0.01 percent to about 1.0 percent.

The present invention provides pharmaceutical compositions, whichcomprise norketotifen formulated together with carefully selectedexcipients. The pharmaceutical compositions of the present inventionsconcern formulations of norketotifen that are intended for topicalophthalmic use by patients suffering from ocular disorders, such asallergic or inflammatory disorders or both allergic and inflammatorydisorders. The terms “composition” and “formulation” are used assynonyms herein. If not stated to the contrary, all percent (%)concentrations in this document refer to percentage by weight (w/w).

The solubility of norketotifen has now been investigated in water and informulations with various excipients. The water solubility ofnorketotifen is low but it was found that the solubility was not furtherdecreased in the formulations of the present invention (Example 10).

The stability of norketotifen formulations has now been investigated.While some excipients significantly decreased the stability, thepreferred formulations that are presented herein, have demonstratedacceptable stability (Example 11).

The therapeutically useful pH-range of solutions of norketotifen waslimited by decreased chemical stability of norketotifen at pH ≧6.5(Example 11). The lowest tolerated pH of ocular formulations is known tobe about pH 4, since formulations with pH <4 may induce chemical burns(Dr Wright Productions, Handbook of Ocular Disease Management, 2009).Thus, the acidity of ocular formulations of norketotifen should be frompH 4 to pH 6.5, preferably from about pH 5.2 to about pH 6.2.

The tonicity of norketotifen formulations should be isotonic to humantears (Benjamin et al., 1983; and Craig e al., 1995.) or slightlyhypotonic. It was therefore determined that the tonicity should be fromabout 150 mOsm to about 450 mOsm, preferably from about 230 mOsm toabout 330 mOsm. As used herein, the term “mOsm” is a measurement ofosmolality and refers to milliosmoles per kilogram of solvent.

The viscosity of norketotifen formulations should be within a range thatfeels comfortable to the patient, while not causing blurring of thevision. Furthermore, the norketotifen formulations should have aviscosity that can be handled easily during manufacturing and filling.It was determined that the norketotifen formulations of the presentinvention should have viscosity of about 1.0 to about 100,000 centipoise(cP), preferably between about 2.0 to about 90,000 cP and mostpreferably from about 2.5 to about 75,000 cP, when tested at roomtemperature. As used herein, the term “cP” indicates a measurement ofviscosity and refers to centipoise (water has the viscosity of 1centipoise at 20° C.). As used herein, the terms “compatible”,“compatibility” and the like, relate to the compatibility between theexcipient(s) and the active ingredient norketotifen.

All compositions intended for use in the eye are required to be sterile.The choice of an appropriate method for sterilization is within thescope of understanding of a person of ordinary skill in the art ofmanufacturing ocular dosage forms. Norketotifen compositions, of thepresent invention, which are stable to increased temperatures, can besterilized by moist heat (autoclaving).

The term autoclaving relates to a standardized thermal heating procedurecharacterized by: Heating a test composition to 120° C. or more for aperiod of 15 minutes or more, wherein said composition is aqueous. Saidaqueous composition is kept in a closed vessel, which vessel istypically a plastic or glass bottle. The pressure during autoclaving istypically 1 bar or more. The autoclaving may preferably range from 120to 150° C., more preferably from 120 to 140° C.; the time needed maypreferably range from 15 to 120 minutes, more preferably from 15 to 60minutes; and the pressure applied may preferably range from 1 to 20 bar,more preferably from 1 to 10 bar, and even more preferably form 1 to 5bar.

Alternatively, ocular norketotifen compositions can be exposure toultraviolet rays or to irradiation, such as gamma irradiation.Formulations can also be processed aseptically, which includesfiltration through sterilizing grade filters, which may have a nominalpore size of 0.22 μm.

Maintaining sterility in multiple-use containers is usually achieved byadding one or more preservatives to the formulations. Alternatively,sterilized single-unit dose packages, such as for example single unitdose vials, ampoules or syringes, containing a sterile norketotifenformulation, as described herein, may be used. However, themanufacturing, handling and distribution of single-unit dose packagesare expensive and the use thereof may be complicated to the patient.

In an embodiment of the present invention, it has now been found thatformulations containing norketotifen are self-preserving (Example 9).The term “self-preserving” as used herein means that said norketotifenformulations do not support microbial growth despite the absence of theaddition of any conventional preservatives in the formulation. The term“formulations do not support microbial growth” means that the number ofinoculated colonies in a formulation remain the same or decline inpreservative challenge tests carried out on said formulation. Aself-preserving formulation of norketotifen will not need apreservative, such as for example BAK, to be included as an excipient insaid formulation. Accordingly, when used in the claims, the term“consisting essentially of” means that any ingredient that if present inthe formulation, would be present in an amount effective to have apreservative effect on the formulation, is excluded.

Alternatively, the self-preserving effects of the present norketotifenformulations may allow the use of very low concentrations ofpreservative agents. A “very low concentration” of the preservativeagent BAK is an amount corresponding to a concentration that is lessthan 0.001 percent by weight of said formulation. A “very lowconcentration” of the preservative agent PHMB is an amount correspondingto a concentration that is equal or less than 1 ppm. Surprisingly, ithas now been found that the anti-microbial effect of norketotifenformulations is pH-dependent; self-preservation was observed innorketotifen formulations at pH 5.0 or higher.

In another embodiment of the present invention, it has now surprisinglybeen found that the preferred formulations of norketotifen prevent theexpression of unwanted ocular effects of norketotifen or anyexcipient(s) being used. Thus, the preferred norketotifen formulationsprevent or inhibit any unwanted effects on the cornea (such as forexample the formation of opacities), on the iris (such as for examplecongestion, swelling or reaction to light) or on the conjunctivae (suchas for example redness, swelling or discharge formation). It has nowsurprisingly been found that certain preferred formulations ofnorketotifen have the ability to completely prevent any and all ocularside effects of norketotifen even at the highest concentrations ofnorketotifen that can be prepared in ocular solutions.

In another embodiment, it has surprisingly been found that the preferredocular formulations of norketotifen do not decrease the solubility ofnorketotifen, but actually increase said solubility. Thus, thesolubility of norketotifen at pH 4.0 was found to be 2.16 mg/ml in waterand the solubility remained approximately the same as pH was increased(by adding NaOH) to pH 5.0, 6.0 or 7.0. The solubility of norketotifenin buffered water was slightly decreased to 2.10 mg/ml at pH 8.6. Thesolubility of norketotifen was sharply decreased at higher pH and at pH8.75 the solubility of norketotifen was decreased to approximately 1.0mg/ml. In the preferred solution formulations NOBAK, LOBAK, MIDBAK,HIBAK, PHMB, S1008, S1009 and S1010 (Tables 1A and 1B), the solubilityof norketotifen was about 2.4 mg/ml (pH 5.5). The solubility onnorketotifen could be significantly increased is certain formulations,such as gels and ointments where solvents were not water. Thus, thesolubility of norketotifen was about 10 mg/ml in propylene glycol.

To avoid ocular irritation by foreign particles, all formulations haveto be foreign particulate free. The term “foreign particulate free”indicates the absence of any particulate matter, but excludes drugparticles, controlled release micro-particulates and the like.

Norketotifen compositions of the present invention can be filled intovials, ampoules, syringes or the like and then lyophilized. Lyophilizedproducts, which are free from moisture, are then reconstituted beforeadministration providing a prolonged shelf life.

The norketotifen formulations for ocular administration that aredescribed herein can be readily processed by standard manufacturingprocesses, well known to those skilled in the art of manufacturingocular dosage forms.

Excipients Compatible with Norketotifen

Suitable excipients, compatible with norketotifen, include specificantioxidants, buffers, chelating agents, emollients, emulsifiers,fillers, gelling agents, humectants, mucoadhesives, preservatives,solvents, stabilizers, surfactants, tonicity agents and viscositymodifying agents. It can be noted that one and the same excipient canbelong to various classes; thus, for example edetate (EDTA) can havebuffering activity, chelating activity, preservative activity,stabilizing activity (also during autoclaving procedures), viscositymodifying activity and possibly additional activities. Another exampleis propylene glycol that is used in formulation S1009 (Table 1B) as asolvent/moisturizer/tonicity modifier.

The term “EDTA”, as used herein, comprises the chemical compoundethylenediaminetetraacetic acid and the disodium and calcium disodiumsalts thereof. EDTA and the salts thereof have many names, such as forexample edetate, disodium edetade. ED3A (ethylenediaminetriacetic acid)may be used instead of or in addition to EDTA in the compositionsdescribed herein.

Antioxidants are compounds that act to slow or prevent the oxidation ofother chemicals. Suitable antioxidants that are compatible withnorketotifen include sulfites, ascorbates, acetylcystein, butylatedhydroxyanisole (BHA) and butylated hydroxytoluene (BHT). When needed,compatible antioxidants can be used in all formulations mentionedherein. Useful concentrations range from about 0.05 percent to about 3percent, preferably 0.1 percent to 0.25 percent, by weight.

Buffering agents are used to adjust the pH of a solution. The functionof a buffering agent is to drive an acidic or alkaline solution to acertain pH range and prevent a change from this pH. Buffering agentshave variable properties—some are more soluble than others; some areacidic while others are basic. Suitable buffering agents that arecompatible with norketotifen include phosphates, boric acid, borates,citrates and acetates. Buffers will be used in the concentrations neededto stabilize the acidity between pH 4.6 and pH 6.5, preferably betweenpH 5.2 and pH 6.2. The amount of each of the buffering compounds neededmay range from about 0.01 percent to about 4 percent by weight,preferably from 0.05 percent to 1 percent by weight. Ocular compositionswith pH ≦4.0 are usually not well accepted by patients and ocularcompositions of norketotifen with pH ≧6.5 have now been found todecrease long-term chemical stability. Thus, the acidity of ocularnorketotifen compositions should be between about pH 4 and about pH 6.5,preferably between pH 5.2 and pH 6.2. When needed, compatible bufferingagents can be used in all formulations mentioned herein. The acidity ofall formulations described herein can be adjusted by changing theconcentrations of the buffering agents or by adding an acid or a base asknown to those skilled in the art.

Chelating agents, which are often organic compounds, are also calledchelants, or sequestering agents and have the ability to form a chelatecomplex with a substrate. Known chelating agents are for example,edetate, proteins, polysaccharides, polynucleic acids and chelatingpolymers. Suitable chelating agents compatible with norketotifen areedetate and chitosan polysaccharides. If needed, chelating agents may beused in concentrations from about 0.01 percent to about 10 percent,preferably from 0.01 percent to 2.0 percent by weight. Some chelatingagents, for example chitosan polysaccharides, also have mucoadhesiveproperties. When needed, compatible chelating agents can be used in allformulations mentioned herein.

Emollients cause occlusion of mucous membranes by providing a layer ofoil to slow water loss from mucous membranes of the eye. Emollients alsoact as humectants and thereby improve the water-holding capacity of theocular tissues. Emollients also act as lubricants, whereby these agentsadd slip or glide to the mucous membranes of the cornea and theconjunctival membranes. Emollients can be used in the ocularformulations of the present invention only if said emollients meet thecriteria of being active at pH ≦6.0 and if they do not decrease thechemical stability of norketotifen. Suitable emollients compatible withnorketotifen include, for example, glycerin, propylene glycol, andhypromellose (hydroxypropyl methylcellulose, HPMC). When needed,compatible emollients can be used in all formulations mentioned herein.If needed, said emollients can be used in concentrations from about 0.1percent to about 10 percent and preferably in concentrations from 0.1percent to 2 percent by weight in the norketotifen formulationsdescribed herein.

Gelling agents (viscosity-modifying agents) are used to thicken andstabilize liquid solutions, emulsions and suspensions, thereby inducingretention of the compositions in the ocular tear fluid. Gelling agentsdissolve in solutions, giving an appearance of a more or less solidmatter, while being mostly composed of a liquid. Examples of suitablegelling agents compatible with norketotifen include edetate (EDTA),alginic acid and alginates, carrageenan, pectin, gelatin and gellingpolymers. When needed, compatible gelling agents can be used in allformulations mentioned herein. Gelling agents can be used inconcentrations from about 0.05 percent to about 10 percent andpreferably in concentrations from 0.1 to 2.5 percent by weight.

In situ gelling agents may be included in ocular formulations ofnorketotifen and are instilled as drops into the eye and undergosol-to-gel transition in the tear fluid, due, for example, toion-triggered activation, pH-triggered activation or thermal activation.Examples: Alginate is a gelling agent that can be used in combinationwith the viscosity-enhancing agent hydroxypropyl methylcellulose (HPMC).The rheological behavior of the alginate/HPMC solutions were retained inthe presence of norketotifen and was found to be a useful ion-activatedin situ gelling system for norketotifen-containing compositions.Polyacrylic acid (Carbopol) is a gelling agent in combination with theviscosity-enhancing agent hydroxypropyl methylcellulose (HPMC) and is auseful pH-triggered in situ gelling system for norketotifen-containingcompositions. Poloxamer 407 is a polymer with a solution viscosity thatincreases when its temperature is raised to the eye temperature (Hongyiet al. 2006). The temperature-sensitive rheological behavior ofPoloxamer 407 or Poloxamer 407/188 mixtures was not influenced by thepresence of norketotifen. Suitable in situ gelling agents compatiblewith norketotifen were also found to include alginate/hydroxypropylmethylcellulose, polyacrylic acid/hydroxypropyl methylcellulose. In situgelling agents as described above can be used in concentrations fromabout 0.5 percent to about 10 percent, preferably from 0.1 percent to2.5 percent by weight. Poloxamers can be used in higher concentrations,up to 25 percent by weight.

Humectants can be used to soften biological tissues as they increase thewater-holding capacity of ocular tissues, such as the cornea and theconjunctival membranes and certain humectants were found to becompatible with norketotifen and can be used in ocular formulations ofnorketotifen. Suitable humectants that are found to be compatible withnorketotifen include polyethylene glycol, sorbitol and propylene glycol.When needed, compatible humectants can be used in all formulationsmentioned herein. Said humectants are used in concentrations from about0.05 percent to about 10 percent, preferably from 0.1 percent to lessthan 4 percent and more preferably from 0.1 percent to 2 percent byweight.

Lubricants can hold moisture on the eye. Numerous polymers can be usedas ocular lubricants. Suitable lubricants that are compatible withnorketotifen include methylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, thiolated acrylic acid polymers, carbomer,carboxymethylcellulose sodium, chitosans, and polyisobutylcyanoacrylate.When needed, compatible lubricants can be used in all formulationsmentioned herein. If needed, the concentrations of said lubricant isfrom 0.1 percent to 10 percent, preferably from about 0.1 percent toabout 4 percent and more preferably from 0.1 percent to 2 percent byweight.

Mucoadhesive agents refer to materials that will adhere to mucus andmucosal membranes. Suitable mucoadhesives that are compatible withnorketotifen formulations described here include thiolated acrylic acidpolymers, chitosan, polyisobutylcyanoacrylate and ethylcellulose.Mucoadhesive polymers, such as mucoadhesive chitosan and mucoadhesivechitosan-coated microspheres or liposomes will be useful for prolongeddelivery of norketotifen to the eye. Mucoadhesive agents can be used inconcentrations from about 0.1 percent to about 10 percent, preferablyfrom 0.1 to 2 percent by weight. If needed, compatible mucoadhesiveagents can be used in all formulations mentioned herein.

Using compatible mucoadhesive agents, norketotifen can be administeredto patients as ocular mucoadhesive minitablets, microspheres and asocular gel-forming minitablets (see Example 7 below).

Preservatives are substances that can be used to prevent the growth ofmicroorganisms in ophthalmic formulations. Suitable preservatives thatare compatible with norketotifen include stabilized oxychloro complexes,benzalkonium chloride (BAK), polyhexamethylene biguanide (PHMB). Sorbicacid was found to be incompatible with norketotifen since thispreservative decreased the stability of norketotifen solutions. Asuitable concentration of a stabilized oxychloro complex is from 0.003percent to 0.01 percent by weight and a suitable concentration of BAK isfrom 0.0001 percent (1 ppm) to 0.05 percent (500 ppm) preferably 0.0001percent to 0.02 percent by weight. A suitable concentration of PHMB isfrom 0.00001 percent (0.1 ppm) to 0.005 percent (50 ppm), preferablyfrom 0.0005 percent (5 ppm) to 0.00005 percent (0.5 ppm). Anypreservative mentioned here may be combined with one or more otherpreservatives for improved efficacy. The concentrations of preservativesmay be kept lower than shown here, including the case where nopreservatives are used, since formulations containing norketotifen havenow been found to be self-preserving.

In addition to water, which is the preferred carrier, other solventslike polyethylene glycol (PEG) and/or propylene glycol (PG) can be usedin ophthalmic compositions. Norketotifen HF was found to be soluble in apolyethylene glycol (PEG 300) up to 0.15 percent w/w. Propylene glycolcan be used as a solvent to obtain high concentrations of norketotifenin ophthalmic ointments and gels since norketotifen HF, has now,surprisingly, been found to be soluble in propylene glycol up to 1.0percent by weight. Norketotifen can be dissolved in water inconcentrations up to 0.216 percent by weight. Suitable non-aqueoussolvents include polyethylene glycol (about 0.1 percent to about 90percent) and propylene glycol (about 0.1 percent to about 90 percent).Both BAK and PHMB were compatible with norketotifen and can be used inall formulations mentioned herein.

Stabilizers in ophthalmic formulations enhance the physical stability ofocular compositions, such as for example emulsions. It was found thatseveral known stabilizers, such as for example xanthan gum and carbomers(acrylic acid polymers), were not compatible with norketotifen sincehazy suspensions were formed. Suitable stabilizers that are compatiblewith norketotifen include methylcellulose, edetate, chitosan,hydroxypropylmethylcellulose and hydroxyethylcellulose. Terms, such as“stabilization”, “stabilizer”, “stability”, when used herein relate tothe stability of the pharmaceutical formulation in total and inparticular to the stability of norketotifen when exposed to storage,oxygen, air, light and or heat (including high-temperaturesterilization, such as autoclavation). The compatible stabilizers listedhere are usually used in concentrations from about 0.05 percent to about4 percent and preferably from 0.05 percent to 2 percent by weight.

Combined stabilizer/solubilizers may be used in formulations containingnorketotifen. Such combined additional stabilizer/solubilizers are forexample cyclodextrins. A preferred cyclodextrin is in particularselected from the group of (α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, hydroxypropyl-β-cyclodextrin,hydroxypropyl-γ-cyclodextrin, dimethyl-β-cyclodextrin anddimethyl-γ-cyclodextrin. The concentrations are generally in the rangeof from about 0.01 percent to about 90 percent, more preferably in therange of from about 0.1 to about 20 percent by weight.

Surfactants reduce the surface tension of liquids, such as for examplewater. Suitable surfactants that are compatible with norketotifeninclude nonionic surfactants, such as for example polysorbates, glycerylstearate, lecithins, polyethoxylated castor oil derivatives andoxyethylated tertiary octylphenol formaldehyde polymers, which incombinations with norketotifen were all found to be less irritating tothe eye than ionic surfactants, which may also be used. If needed,compatible surfactants can be used in all formulations mentioned herein.Surfactants are usually used in concentrations from about 0.05 percentto about 4 percent and preferably from 0.1 percent to 2 percent byweight.

Tonicity-adjusting agents increase the effective osmolarity or effectiveosmolality of a formulation. Hypertonic, hypotonic and isotonicsolutions are defined in reference to a cell membrane by comparing thetonicity of the solution with the tonicity within the cell. Ocularcompositions preferably contain a tonicity-adjusting agent in an amountsufficient to cause the final composition to have an ophthalmicallyacceptable osmolality (generally 150 to 450 mOsm and preferably 230 to330 mOsm). Suitable tonicity-adjusting agents to be used withnorketotifen may be of ionic and/or non-ionic type. An example of ionictype tonicity enhancers is sodium chloride and examples of non-ionictonicity enhancing agents are, for example sorbitol and propyleneglycol, which are compatible with norketotifen. Thus, norketotifenformulations may include for example sodium chloride in concentrationsfrom about 0.1 to about 0.9 percent by weight, sorbitol inconcentrations from about 0.1 to about 10 percent or propylene glycol inconcentrations from about 0.1 to about 10 percent by weight. If needed,compatible tonicity-adjusting agents can be used in all formulationsmentioned herein. All ophthalmic formulations of norketotifen wereadjusted to be approximately iso-osmotic to human tears (Benjamin etal., 1983; Craig et al., 1995.)

Viscosity-adjusting agents increase the internal friction (“thickness”)of a formulation. The ophthalmic solutions of the present invention maycontain one or more viscosity-adjusting agent and have a viscosity of1.0 to 100,000 cP, preferably between 2.0 to 90,000 cP, and mostpreferred between 2.5 and 75,000 cP, which is acceptable sincecompositions in this range of viscosity feel comfortable to the eye anddo not cause blurring of the vision. Viscosity modifying agents can beused in ophthalmic compositions and are substances that have the abilityto cause thickening (increase the viscosity) of ophthalmic formulations.Viscosified solutions are accepted to a great degree by patients, mainlybecause of the ease of administration. Some viscosity modifying agents,such as for example xanthan gum, were not compatible with norketotifen.Viscosity modifying agents that are compatible with norketotifen includeedetate, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, polyethylene glycol, propyleneglycol alginate, chitosan, and tragacanth. The term “hydrogels” is oftenused for viscosity enhancing excipients, particularly in artificialtears and refers to a colloid with high gelling ability. If needed,compatible viscosity-adjusting agents can be used in all formulationsmentioned herein. When needed, the concentrations of the selectedviscosity modifying agents range from about 0.1 percent to about 10percent by weight, and preferably between 1 percent and 5 percent.Sorbitol may be used as a combined tonicity-adjusting andviscosity-adjusting excipient in a concentration range from about 0.1 toabout 10 percent, preferably from 2 percent to 5 percent.

There are currently two strategies to increase the retention time ofocular formulations in the eye. Thus, either excipients can be used thathave bioadhesive properties, such as for example mucoadhesiveexcipients, or the formulation can be made more viscous. Both strategiesare included in the present invention.

In certain embodiments, the compositions containing norketotifen arepacked in opaque plastic containers that may be sterilized using forexample ethylene oxide or gamma radiation. A preferred container for anophthalmic product may be equipped with an eyedropper. Single-dosecontainers may be used and have advantages that are obvious to thoseskilled in the art.

Compositions Compatible with Norketotifen

Using excipients that had been found to be compatible with norketotifen,compositions such as topical ophthalmic solutions, topical ophthalmicgels, topical hydrophilic ophthalmic ointments, topical ophthalmicemulsions, and topical ophthalmic liposome compositions were prepared asdemonstrated in the following Examples. The prepared formulations weretested for physical appearance and stability (refrigerated, roomtemperature, and at increased temperatures) using analytical methodologyas described in Example 10.

EXAMPLES

Certain embodiments of the present invention are illustrated in thefollowing examples. The embodiments described in this specification areconsidered to be illustrative in all respects and not restrictive. Thescope of this invention is indicated by the appended claims, not by thisdescription.

An HPLC method for the determination of concentrations of norketotifenwas developed (Example 10). The excipients used in the presentcompositions can be analyzed using standard methods that are well knownto those skilled in the art.

Example 1 Ophthalmic Solutions

Examples of preferred solution formulations containing norketotifenhydrogen fumarate are shown in Tables 1A and 1B.

Preferred solution formulations containing norketotifen may containexcipients at different concentrations from those shown in Tables 1A and1B.

Other useful solution formulation containing norketotifen may containexcipients that are different from those shown in Tables 1A and 1B.

TABLE 1A Examples of preferred solution formulations containingnorketotifen HF. Excipients in % NOBAK LOBAK MIDBAK HIBAK PHMBNorketotifen 0.0345 (1)   0.0345 0.0345 (1) 0.0345 (1) 0.0345 (1) HF (*)(%) (1) EDTA 0.100   0.100 0.100 0.100 0.100 Boric Acid 0.095   0.0950.095 0.095 0.095 BAK — ≦0.001 0.005 0.010 — (**) PHMB — — — — 0.0001Sorbitol 4.600   4.600 4.600 4.600 4.600 Water q.s. q.s. q.s. q.s. q.s.pH (***) 4.6-6.2   4.6-6.2 4.6-6.2 4.6-6.2 4.6-6.2 (1) Norketotifen HF0.0345 percent is equivalent to norketotifen FB 0.025 percent. (*)Norketotifen may be used in concentrations from 0.01 to 0.3 percent.(**) BAK-concentration is from 0.0001% to 0.001%, preferably 0.0005%(***) pH is between 4.6 and 6.2, preferably 5.5.

TABLE 1B Examples of preferred solution formulations containingnorketotifen. Excipients in percent S1008 S1009 S1010 Norketotifen HF(%) (*) 0.0345 (1) 0.208 (2) 0.208 (2) Sodium phosphate dibasic 0.473 —0.160 Sodium phosphate•monobasic, 0.460 — — monohydrate NaCl 0.480 — —BAK 0.010 — 0.010 Sodium citrate — 0.300 — Propylene glycol — 1.750 —Methylparaben — 0.030 — Propylparaben — 0.010 — Methylcellulose — —0.500 Glycerin — — 2.400 Water q.s. q.s. q.s. pH (**) 4.6-6.2 4.6-6.24.6-6.2 (1) Norketotifen HF 0.0345 percent is equivalent to 0.025percent norketotifen FB. (2) Norketotifen HF 0.208 percent is equivalentto 0.15 percent norketotifen FB. (*) Norketotifen HF may be used inconcentrations from 0.01% to 0.3%. (**) pH is between 4.6 and 6.2,preferably adjusted to 5.5.

All ophthalmic formulations of norketotifen were adjusted to beapproximately iso-osmotic to human tears (Benjamin et al., 1983; Craiget al., 1995.)

If needed, the tonicity can be adjusted by adding a tonicity-adjustingagent to obtain the preferred tonicity. If needed, the viscosity can beadjusted by a viscosity-modifying agent to obtain the preferredviscosity. The final acidity can be adjusted by adjusting theconcentrations of the buffering agents or by adding an acid or a base.

The solution formulations were prepared by adding the excipients, one ata time to an appropriate amount of water, followed by mixing untildissolved. Once all excipients had been added and dissolved,norketotifen was added to the solution of excipients and mixedcontinuously until dissolved. The acidity of the solutions was measuredand adjusted by modifying the buffer system or by adding an acid or abase solution to the desired pH. If needed, viscosity and tonicity wereadjusted as indicated above.

Example 2 Ophthalmic Ointments and Gels

An example of a preferred composition for a topical hydrophilicophthalmic gel containing norketotifen hydrogen fumarate (HF) is shownin Table 2.

Preferred ophthalmic hydrophilic ointments or gels containingnorketotifen may contain excipients at concentrations that are differentfrom those shown in Table 2. Ophthalmic hydrophilic ointments or gelscontaining norketotifen may contain excipients that are different fromthose shown in Table 2.

Topical hydrophilic ophthalmic gel and ointment compositions containingnorketotifen can keep the drug in the eye for an extended period of timeand the prolonged exposure will enhance drug delivery.

Ophthalmic hydrophilic ointments and gels were made, comprisingnorketotifen at concentrations that were usually between 0.010 percentand 1.0 percent (w/w, calculated as free base), although suchformulations may contain in excess of 5.0 percent of norketotifen. Saidhydrophilic ointment and gel formulations have a viscosity that rangedfrom 5,000 to 500,000 cP, preferably from 20,000 to 200,000 cP. Examplesof thickeners/gelling agents, used in the present studies, arepolyethylene glycol 300 and/or polyethylene glycol 3350 and/orpolyethylene sorbate (polysorbate) and/or chitosan. A compatiblesurfactant, such as poloxamer 407 can also be added, preferably in aconcentration less than 25 percent, more preferred in a concentrationless than 20 percent by weight. It was also found that ophthalmichydrophilic ointments and gels, containing norketotifen, could alsocontain selected excipients, such as humectants such as for examplesorbitol, viscosity modifying agents such as for example methylcellulose, tonicity agents such as for example NaCl or propylene glycol,chelating agents such as for example edetate or polysaccharides, bufferssuch as for example phosphate buffers, surfactants such as for exampleglyceryl stearate, mucoadhesives such as for examplepolyisobutylcyanoacrylate, antioxidants such as for example BHA or BHTand preservatives such as for example BAK or PHMB. Suggestedconcentrations of these excipients are as shown previously in thisdocument.

Said gels and ophthalmic hydrophilic ointments were designed foronce-daily ocular administration or for repeated administrations fromtwo to five times daily. The terms “gel” and “ointment” are usedinterchangeably.

The selected hydrophilic ointment/gel in Table 2 is thick but misciblewith water. This composition can hold the drug product in the eye of thepatient for an extended time, which will enhance drug delivery.

TABLE 2 An example of a preferred topical hydrophilic ophthalmicointment or gel containing norketotifen. Batch OG1009 Norketotifenhydrogen fumarate (%) 0.208 (1) or Norketotifen free base (%) 0.150 PEG300 (%) if norketotifen HF is used 69.790 PEG 300 (%) if norketotifen FBis used 69.850 PEG 3350 (%) 30.000 (1) Equivalent to 0.15 percent ofnorketotifen free base (FB)

Batch OG1009 used a mixture of the polyethylene glycols PEG 300 and PEG3350 as solvent for norketotifen.

The composition of Table 2 was prepared by adding the two polyethyleneglycols to a suitable container and heating to 60-65° C. This heatingstep melts the high molecular weight polyethyleneglycol. Next,norketotifen was added and the composition was mixed until the activeingredient was dissolved. Finally, the composition was cooled withmixing to allow the ointment/gel to thicken. The viscosity was 30,000 cPor greater. The pH range for these compositions was not measured sincethe formulations were non-aqueous. If needed, the tonicity can beadjusted by adding a tonicity-adjusting agent to obtain the preferredtonicity. If needed, a preservative can be added. The concentration ofnorketotifen HF can be from about 0.001% to about 0.15% by weight ifdissolved in PEG 300 and from about 0.001% to about 0.25% in a mixtureof PEG 300 and PEG 3350.

Example 3 Ophthalmic Hydrophobic Ointments

An example of preferred compositions for topical hydrophobic ophthalmicointments containing norketotifen hydrogen fumarate (HF) is shown inTable 3.

Preferred hydrophobic ophthalmic ointments containing norketotifen maycontain excipients at concentrations that are different from those shownin Table 3. Hydrophobic ophthalmic ointments containing norketotifen maycontain excipients that are different from those shown in Table 3.

The tested hydrophobic ointments were not miscible with water. Thesecompositions can hold the drug product in the eye of the patient for anextended time and will enhance drug delivery.

Ophthalmic hydrophobic ointments and gels may contain norketotifen atconcentrations between 0.001 percent and 5.0 percent, more preferablybetween 0.01 percent and 1.0 percent. Said ophthalmic hydrophobicointments and gel solutions were having viscosity in the range of rangefrom 5,000 to 500,000 cP and preferably from 20,000 to 200,000 cP. Saidophthalmic hydrophobic ointments and gels have tonicity between 150 and450 mOsm, preferably between 230 and 330 mOsm. Said ophthalmichydrophobic ointments and gels can also contain other excipients, suchas humectants, viscosity modifying agents, tonicity agents, chelatingagents, buffers, surfactants, mucoadhesives, antioxidants andpreservatives. Said ophthalmic hydrophobic ointments and gels weredesigned for once-daily ocular administration or for repeated ocularadministrations from two to five times daily to a mammal in needthereof.

TABLE 3 An example of a preferred hydrophobic ointments containingnorketotifen HF. Batch HO1012 Norketotifen HF (%) 0.0346 (1) Propyleneglycol (%) 2.500 Glyceryl stearate (%) 0.500 Cetyl alcohol (%) 0.500White petrolatum q.s. (2) (1) Equivalent to 0.025 percent w/w as freebase. (2) quantum sufficit

Batch HO1012 contained propylene glycol as a solvent for norketotifen,glycerol stearate and cetyl alcohol as surfactants and white petrolatumas base.

The hydrophobic ointment was prepared by dissolving norketotifen inpropylene glycol. Next, glyceryl stearate, cetyl alcohol, and whitepetrolatum were added to a suitable container and heated to 65-70° C.This heating step melts the surfactants and the petrolatum. Next,norketotifen solution was slowly added and the composition mixed untilthe solvent was dispersed. Finally, the composition was cooled withmixing to allow the ointment to thicken.

If needed, acidity can be adjusted by adding an acid solution or a basesolution to obtain the preferred acidity. If needed, tonicity can beadjusted by adding a tonicity-adjusting agent to obtain the preferredtonicity. If needed, viscosity can be adjusted by a viscosity-modifyingagent to obtain the preferred viscosity. If needed, a preservative canbe added.

Example 4 Ophthalmic Emulsions

Two examples of preferred compositions for topical ophthalmic emulsionscontaining norketotifen hydrogen fumarate (HF) are shown in Table 4below (E1012 and E1015). Batches E1112 and E1115 were identical to E1012and E1015, but were without the preservatives.

Topical ophthalmic emulsions containing norketotifen may containexcipients that are different from those shown in the examples in Table4.

Ophthalmic emulsions were made, comprising norketotifen atconcentrations between 0.01 percent and 5.0 percent, preferably between0.01 percent and 1.0 percent. Said ophthalmic emulsions were having aviscosity in the range from 1.0 to 300,000 cP and preferably from 2.0 to90,000 cP and most preferred from 2.5 to 75,000 cP. Said ophthalmicemulsions had osmolality between 150 and 450 mOsm and most preferablybetween 230 and 330 mOsm. Said ophthalmic emulsions had pH of 4 to 7,preferably pH 5.2 to 6.2. Said ophthalmic emulsions could also containexcipients, such as humectants, viscosity modifying agents, tonicityagents, chelating agents, buffers, surfactants, mucoadhesives,antioxidants and preservatives.

TABLE 4 Examples of preferred ophthalmic emulsions containingnorketotifen. All composition values are concentrations in percent w/w.Excipients in percent E1012 E1015 E1112 E1115 Norketotifen HF (%) 0.208(1) 0.208 (1) 0.208 (1) 0.208 (1) Sodium phosphate 0.160 0.160 0.1600.160 dibasic Propylene glycol 1.850 1.850 1.850 1.850 Methylparaben0.050 0.050 — — Propylparaben 0.010 0.010 — — Castor oil 1.250 1.2501.250 1.250 Polyoxyl 35 castor oil 1.000 1.000 1.000 1.000Methylcellulose 0.200 — 0.200 — 1.0 N HCl or q.s. to q.s. to q.s. toq.s. to 1.0 N NaOH target pH target pH target pH target pH Water q.s.(2) q.s. (2) q.s. (2) q.s. (2) pH 5.5  5.5  5.5  5.5  (1) Equivalent to0.15 percent as free base; (2) quantum sufficit

The batches E1012 and E1015 contained a phosphate buffer, propyleneglycol as solvent/moisturizer/tonicity modifier, parabens aspreservatives, castor oil and polyoxyl castor oil as surfactants andmethylcellulose (if used) as a stabilizer/viscosity modifier. BatchesE1112 and E1115 were identical to E1012 and E1015, but were without thepreservatives. Several experiments were done and acidity was adjustedover a wide range. It was determined that emulsions can be used at pH ofabout 5.0 to about 7.0; the preferred acidity is pH 5.5 to pH 6.0.

The selected emulsions in Table 4 were prepared by adding propyleneglycol, parabens (if used), castor oil, ethoxylated castor oil, andwater to a suitable container. The contents of the container weresonicated with a 1/2″ ultrasonic probe (Sonics Inc. Vibra Cell) for 20minutes. The resulting emulsion droplets were mostly less than 0.5microns. The emulsion was filtered through a 0.22-micron celluloseacetate filter. After filtration, norketotifen, buffer salt, and polymer(if used) were added.

If needed, the tonicity can be adjusted by adding a tonicity-adjustingagent to obtain the preferred tonicity. The viscosity of norketotifenemulsions can be adjusted by a compatible viscosity-modifying agent inan amount that is needed to obtain the preferred viscosity.

Example 5 Ophthalmic Liposome Compositions

An example of a preferred ophthalmic liposome composition containingnorketotifen hydrogen fumarate (HF) is shown in table 5. Preferredophthalmic liposome compositions containing norketotifen may containexcipients of concentrations that are different from those shown inTable 5. Ophthalmic liposome compositions containing norketotifen maycontain excipients that are different from those shown in Table 5.

Ophthalmic liposome compositions were made, comprising norketotifen atconcentrations preferably between 0.01 percent and 0.50 percent. Saidophthalmic liposome compositions were having a preferred viscosity thatranged from 1.0 to 100,000 cP and more preferably from 2.0 to 90,000 cP.Said ophthalmic liposome compositions were having an osmolality between150 and 450 mOsm, preferably between 230 and 330 mOsm. Ophthalmicliposome compositions have pH of 4 to 7, preferably pH 5.2 to 6.2. Theliposome compositions are approximately iso-osmotic. Said ophthalmicemulsions also contained excipients, such as humectants, viscositymodifying agents, tonicity agents, chelating agents, buffers,surfactants, muco-adhesives, antioxidants and preservatives. Saidophthalmic emulsions were designed for once-daily ocular administrationor for repeated ocular administrations from two to five times daily to amammal in need thereof.

Incorporating norketotifen in a selected liposome composition willenhance residence time in the eye and improve ocular drug delivery tothe tissues. A preferred liposome composition containing norketotifen isshown in Table 5 below. The acidity can be changed by adjustment of thebuffer or by adding an acid or a base as know as known to those skilledin the art.

TABLE 5 Example of a preferred ophthalmic liposome compositioncontaining norketotifen HF. Excipients in percent LIP1011 NorketotifenHF (%) 0.208 (1) Sodium phosphate dibasic 0.160 Glycerin 2.400Benzalkonium chloride 0.010 Soy lecithin 1.000 Cholesterol 0.050 Waterq.s. (2) 1.0 N HCl q.s. to target pH pH 4.6 to 6.2 (1) Equivalent to0.15 percent w/w as free base; (2) quantum sufficit pH is 4.6 to 6.2,preferably about 5.5.

Batch LIP1011 contained dibasic sodium phosphate as a phosphate buffer,glycerin as a moisturizer/tonicity modifier, benzalkonium chloride as apreservative and soy lecithin and cholesterol as surfactants. Severalexperiments were done and acidity was adjusted over a wide range. It wasdetermined that emulsions can be used at an acidity from about pH 5.0 toabout pH 7.0; the preferred acidity is pH 4.6 to pH 6.2; most preferredis pH 5.5.

To prepare liposome compositions containing norketotifen, lecithin andcholesterol were dissolved in ethanol and added to a suitable container.Ethanol was evaporated leaving behind a lipid film. The remainingcomponents of the composition were dissolved in water and this solutionwas added to the lipids to form a coarse dispersion of vesicles. Thisdispersion was sonicated with a ½″ ultrasonic probe (Sonics Inc. VibraCell) for 15 minutes. The resulting liposomes were mostly less than 0.5microns. The liposome composition was filtered through a 0.22-microncellulose acetate filter.

If needed, the tonicity can be adjusted by adding a tonicity-adjustingagent to obtain the preferred tonicity. If needed, the viscosity can beadjusted by a viscosity-modifying agent to obtain the preferredviscosity.

Example 6 Ophthalmic Solutions of Norketotifen Free Base

Ophthalmic compositions containing norketotifen free base may containexcipients that are different from those shown in Tables 1A, 1B or 7.

Compositions containing solutions of norketotifen FB represent achallenge due to the low water solubility of the free base. It has nowbeen found that norketotifen FB can be dissolved in propylene glycol,glycerin, ethanol and isopropyl alcohol. Surfactants, such as forexample polysorbate 80, polysorbate 20, poloxamer 407, poloxamer 188,polyoxyl 40 stearate or sorbitan monolaurate can also be used todissolve norketotifen FB. Liposomes, as for example those made withphospholipids can also be used with the free base of norketotifen.Norketotifen FB can also be formulated as a suspension and otherformulations of the present invention. Several experiments were done andacidity was adjusted over a wide range. It was determined that emulsionscan be used at pH of about 5.0 to about 7.0; the preferred acidity is pH5.5. An example of a preferred suspension containing norketotifen freebase is shown in Table 6, below, where all percent are w/w.

TABLE 6 An example of a preferred ophthalmic suspension, containingnorketotifen free base (FB). SUS1011 Norketotifen FB (%)  0.20 Poloxamer407 (%) 0.2 Boric acid (%) 0.1 Sodium chloride (%)  0.09 HCl/NaOH for pHAs needed adjustment Water q.s. (1) pH 5.5 (1) quantum sufficit

If needed, the tonicity can be adjusted by adding a tonicity-adjustingagent to obtain the preferred tonicity. If needed, the viscosity can beadjusted by a viscosity-modifying agent to obtain the preferredviscosity. If needed, a preservative, such as for example BAK may beadded in concentrations shown previously in this document.

Example 7 Ocular Minitablets

Use of Ocular Mucoadhesive Minitablets and Gel-Forming Minitablets

Mucoadhesive Ocular Minitablets

Norketotifen ocular minitablets, using pregelatinized starch,hydroxypropylcellulose and Avicel and containing 5 percent Carbopol asthe bioadhesive polymer (to reduce the risk for expulsion) and one ormore water-insoluble polymers, such as ethylcellulose, unmodified orthiolated polyacrylic acid are prepared by compression. Minitablets areplaced under the eyelid of rabbits. The effects on tear-film stabilityare examined using fluorescein staining after single dosing. Preliminaryresults indicate that inserts containing thiolated polyacrylic acidoffer prolonged fluorescein concentration on the eye.

Gel-Forming Ocular Minitablets

Gel-forming ocular minitablets made of thiomers may also be used as anocular dosage form for norketotifen.

Example 8 Ocular Irritation Test

Tests of Ocular Irritation of Norketotifen in Various Formulations.

Methods

New Zealand White rabbits, weighing 2 to 4 kilograms were used. Testformulation, in volumes of 0.1 ml were instilled into the conjunctivalsac of six rabbit eyes of groups. Said volume of each formulation wasrepeatedly instilled every 30 minutes for 3 hours (a total of seveninstillation into the eyes of rabbits. Ocular irritation was scoredaccording to both Draize et al., 1944 and Kay and Calandra, 1962.Scorings were performed before each instillation and 30 minutes, 3 hoursand 24 hours after the last instillation.

The formulations of norketotifen in Table 7 were prepared and tested andthe results were compared with irritation by norketotifen inexcipient-free solutions. Table 7 indicates that the solutions ofnorketotifen HF were 0.276% since the solubility of norketotifenhydrogen fumarate (HF) in some or all of these solutions may have beenless than 0.276 percent. In the present study, norketotifen HF wasdissolved to an intended concentration of 0.276 percent in the varioussolutions, using stirring and over-night sonication at room temperature.After sonication, all solutions were filtered through a 0.45 μmnon-pyrogenic MILLEX® HV filter unit before being used for ocular tests.Thus, all of the solutions used for the ocular irritation tests in Table7 contained the highest possible concentrations of norketotifen HF.

TABLE 7 Formulations used for ocular irritation tests IngredientsFormulation A Formulation B Formulation C Formulation D Norketotifen HF(*) ≦0.276%    ≦0.276%    ≦0.276%    ≦0.276%    Sodium phosphate 0.473%— — — dibasic Sodium phosphate 0.460% — — — monobasic, monohydrateSodium Chloride 0.480% — — — Benzalkonium chloride 0.010% — 0.010% —(BAK) Sodium citrate — 0.300% — — Propylene glycol — 1.750% — —Methylparaben — 0.030% — — Propylparaben — 0.010% — — EDTA — — 0.100%0.100% Boric Acid — — 0.095% 0.095% Sorbitol — —  4.6%  4.6% Water q.s.q.s. q.s. q.s. pH 5.5 5.5 5.5 5.5 (*) 0.276% norketotifen HF = 0.20%norketotifen FB.

Solutions of norketotifen were similarly prepared in excipient-freesolutions containing only norketotifen HF 0.0345 percent by weight,dissolved in water or in saline, and filtered as described above. Theexcipient-free solutions containing norketotifen HF were tested byocular instillations in one eye of 6 rabbits, followed by repeatedDraize scorings and Kay & Calandra scorings as described above.

Results

The irritation scoring demonstrated that excipient-free norketotifen HF,even at the low concentration of 0.0345% caused irritation afterinstillations into rabbit eyes. No ocular irritation by norketotifen wasobserved when norketotifen in the highest possible concentration(≦0.276%) was administered in either of the Formulations A, B, C, or D.

Conclusions

It was surprisingly found that even in the highest concentrationspossible, and even after seven consecutive instillations into the eye,norketotifen was not causing any ocular irritation, when administered inthe preferred formulations, herein represented by Formulations A, B, Cand D. These results were contrary to results obtained withexcipient-free solutions.

Example 9 Antimicrobial Effectiveness Testing

Preservative Challenge Tests of New Formulations

Ocular products in multidose containers must be adequately preserved toprevent contamination during repeated use. The most commonly usedpreservative, benzalkonium chloride (BAK) has known side effects on theeye (Baudouin, 2008) and efforts are made to decrease the concentrationof BAK (Table 1A: LOBAK formulations) or completely eliminate BAK (Table1A: NOBAK formulations).

Method

Preservative challenge tests, slightly modified after U.S. Pharmacopeia51 Antimicrobial Effectiveness Testing, were performed. USP (51) is astandard test used to determine the effectiveness of antimicrobialsubstances in ophthalmic products. The present tests were using 150 mlof each formulation and covered five organisms: Escherichia coli(fermentative G−), Pseudomonas aeruginosa (non-fermentative G−),Staphylococcus aureus (G+), Aspergillus niger (fungus) and Candidaalbicans (yeast).

The number of inoculated cells was 0.5×10⁵ per mL and the incubationtemperature was 25° C. (rt) for all inoculations. Plating for measuringof recoveries were performed weekly over 4 weeks. If the numbers of cellcolonies remained constant or were declining for all fivemicroorganisms, the norketotifen formulation was defined as havingself-preserving qualities; if the numbers of colonies increased for anyof the five microorganisms, the formulations were not considered ashaving self-preserving activity.

Tests were performed at pH 4.0, 4.5, pH 5.0 and pH 6.0 and withformulations containing 0.208 percent norketotifen HF. All formulationsreferred to here were devoid of preservatives and contained: EDTA 0.1%;Boric acid 0.095%; Sorbitol 4.6% and water q.s. (see Table 1A: NOBAKformulations).

Results

The antimicrobial activity of the norketotifen formulations waspH-dependent. When tested at pH 5 and pH 6, preferred formulationscontaining 0.208 percent or 0.0345 percent norketotifen HF weresurprisingly found to be self-preserving. Thus, the numbers of coloniesof all five microorganisms declined or stayed the same during the courseof the 4-week tests in formulation with pH ≧5.0.

TABLE 8 Results from preservative challenge tests of formulationscontaining norketotifen 0.0345 percent (NOBAK formulation; Table 1A).FORMULATION pH 4.0 pH 4.5 pH 5.0 pH 6.0 NOBAK *** **** ***** ***** ***indicate that for three of the five microorganisms the numbers ofcolonies declined or remained constant (the formulation did not pass thetest). **** indicate that for four of the five microorganisms thenumbers of colonies declined or remained constant (the formulation didnot pass the test). ***** indicate that for all five microorganisms thenumbers of colonies declined or remained constant (the formulationpassed the test).Conclusion

Ocular formulations of norketotifen according to the present inventioncan be used without preservatives in formulation with pH ≧5.0.

Example 10 Solubility of Norketotifen

The norketotifen hydrogen fumarate salt and other acceptable salt forms,such as for example the hydrochloride salt are acid salts of a weakbase. As such the salts can be expected to have fairly constantsolubility over a wide pH range. Formulations that contain an excipientor a combination of excipients that decrease the solubility of theactive ingredient may not be useful.

Method

Saturated solutions of norketotifen were prepared at room temperature bystirring, overnight sonication and filtering of the saturatedsupernatant solutions. An HPLC method for the determination ofconcentrations of norketotifen was developed:

Column XTerra RP-C₁₈ (4.6 × 150 mm, 5 mm) Detection UV at 300 nm Mobilephase A: 10% ACN/90% H₂O, 0.1% TFA B: 90% t ACN/10% H₂O, 0.1% TFAGradient 0.0 min.: 100% A; 15.0 min.: 100% B; 15.5 min.: 100% A. Runtime: 25 min Flow rate 1 mL Column Temp. 30° C. Sample conc. 0.025 to0.1 mg/mL Injection volume 20 mL ACN = acetonitrile TFA =trifluoroacetic acid

The HPLC system was a HP 1050 with a variable wavelength detector. Overa range of 0.025 to 0.1 mg/mL, the assay showed good linearity with acorrelation coefficient greater than 0.999. The relative standarddeviation was approximately 0.3 percent for the main peak area.

The HPLC assay described here was also used for determination ofconcentrations of norketotifen in the compositions of the presentinvention. Prior to analysis, norketotifen was extracted from theophthalmic compositions, using standard methods, well known to thoseskilled in analytical chemistry.

Results

The solubility of norketotifen at pH 4.0 was found to be 2.16 mg/ml in(unbuffered) water and the solubility remained approximately the same aspH was increased (by adding NaOH) to pH 5.0, pH 6.0 and pH 7.0. Thewater solubility of norketotifen was decreased to 2.10 mg/ml at pH 8.6.The water solubility of norketotifen was sharply decreased at higher pHand at pH 8.75 the solubility of norketotifen was decreased toapproximately 1.0 mg/ml.

In the preferred solution formulations NOBAK, LOBAK, MIDBAK, HIBAK,PHMB, S1008, S1009 and S1010 (Tables 1A and 1B), the solubility ofnorketotifen was from about 2.4 mg/ml to about 2.7 mg/ml (pH 5.5),representing an improved solubility when compared with solubility inwater.

The solubility of norketotifen was significantly increased in certainformulations, such as gels and ointments where solvents were not water.Thus, the solubility of norketotifen was about 10 mg/ml in formulationOG1009 (Table 2), where the solvent was a mixture of two polyethyleneglycols.

Example 11 Stability of Norketotifen Formulations

Stability of norketotifen formulations was studied after incubation atvarious temperatures, acidity and time intervals.

Methods

The concentrations of norketotifen were measured after incubationperiods of various lengths.

Results

Examples of test results are shown Table 9. All concentrations ofnorketotifen are mg/ml.

TABLE 9A Stability of a formulation containing benzalkonium chloride.Formulation (See Table 1A): LOBAK; pH 5.5. Concentrations are in mg/ml.Temperature Week 1 Week 2 Week 3 Week 4  5° C. 0.3454 0.3438 0.34230.3442 25° C. 0.3453 0.3424 0.3439 0.3426 40° C. 0.3453 0.3425 0.34370.3444 55° C. 0.3430 0.3391 0.3449 0.3462

TABLE 9B Stability of a norketotifen formulation without benzalkoniumchloride. Formulation (See Table 1A): NOBAK; pH 5.5. Concentrations arein mg/ml. Temperature Week 1 Week 2 Week 3 Week 4  5° C. 0.3429 0.34210.3432 0.3442 25° C. 0.3436 0.3426 0.3426 0.3438 40° C. 0.3429 0.34190.3435 0.3457 55° C. 0.3412 0.3416 0.3400 0.3440

TABLE 9C Stability of a norketotifen formulation without benzalkoniumchloride. Formulation (See Table 1A): NOBAK; pH 6.5. Concentrations arein mg/ml. Temperature Week 1 Week 2 Week 3 Week 4  5° C. 0.3436 0.34090.3429 0.3422 25° C. 0.3417 0.3400 0.3408 0.3433 40° C. 0.3397 0.33750.3383 0.3371 55° C. 0.3199 0.3075 0.3014 0.2853Conclusions

It can be concluded from Tables 9A and 9B that the formulations LOBAKand NOBAK at pH 5.5 are stable over a prolonged period as substantiatedby the results from the accelerated stability tests at 40° C. and 55° C.

Table 9C demonstrates that norketotifen is not stable in solutionformulations of pH 6.5 under conditions of accelerated testing (55° C.).

Example 12 Antihistaminic Activity

Antihistaminic (H-1) Activity In Vivo after Oral Administrations toRats.

Methods

Male rats (150-200 g) were starved overnight and twelve hours afterdorsal depilation, the animals were dosed orally with the testcompound(s). Four dorsal test areas were marked with permanent ink.Exactly 60 min after the dosing of the test compound, two intradermalinjections of histamine (50 microliter; 1.0 mg/ml of histamine di-HCl)were performed, one on each side on the back of the animal. Twointradermal injections of the vehicle for the histamine solution werealso performed. Evans blue dye (20 mg/kg) was injected iv 1 minute priorto the intra-dermal injections of histamine and the histamine vehicle.Twenty minutes were allowed for the wheal response to fully develop,whereupon the animals were euthanized and the dorsal skin containing theintradermal wheals was deflected. The blue spotted areas were measuredin square millimeters and the duplicate vehicle wheal responses wereaveraged. In vehicle-treated animals, the wheal area, on average, wasincreased by histamine by 98 mm². The inhibition was calculated inpercent difference from the corresponding vehicle baseline value.

Results

TABLE 10 Antihistaminic activity in vivo after oral administration. Testcompound/ Histamine Saline Histamine effect Inhibition Dose (mg/kg)(mm²) (mm²) (mm²) (percent) Vehicle* 122 ± 9  24 ± 2 98 — Vehicle** 107± 4  25 ± 1 82 — Ketotifen; 1 68 ± 6 21 ± 2 47 43 Ketotifen; 10 24 ± 222 ± 3 2 97 Norketotifen; 1 114 ± 8  22 ± 1 92 6 Norketotifen; 10 39 ± 222 ± 1 17 83 Norketotifen; 50 10 ± 1 12 ± 1 0 100 DPH; 10*** 31 *Vehiclefor norketotifen. **Vehicle for ketotifen. ***DPH = diphenhydramine(generic)

Both ketotifen and norketotifen are potent antihistaminic compounds. Thedose/response curves for ketotifen and norketotifen were not parallel.Ketotifen was approximately twice as potent as norketotifen as anantihistamine at dose levels offering 75% inhibition of the histamineeffect (IC₇₅). Both ketotifen and norketotifen were more potent than thereference compound (diphenhydramine).

Conclusions

As an antihistamine, ketotifen was more potent than norketotifen andboth compounds were more potent than diphenhydramine (Benadryl®). It isalso concluded that norketotifen was well absorbed in rats after oraladministration.

Example 13 Inhibition of Granulocyte Activation

Anti-inflammatory activity was measured as effects on granulocyteactivation (“mast cell stabilization”).

Methods

In the present studies, effects on granulocyte activation was studied asinhibition of histamine release from human granulocytes (buffy coat) bynorketotifen. The method is a modification of the method described byNolte, H. et al., 1988. Granulocytes were obtained from human volunteersand mediator release was induced by incubation (20 min/37° C.) with thecalcium ionophore A23187 (5 μM) in the presence or absence of a testarticle. Histamine was selected as an indicator for mediator releasebecause of the ease with which histamine can be analyzed, usingcommercially available kits. The test articles were evaluated, induplicate, at five concentrations and IC50-values were calculated.

The calcium ionophore A23187 was used to increase intracellular calciumconcentration of the granulocytes. Those skilled in the art ofpharmacology realize that the presently used A23187-method mimics invivo activation, initiated by IgE- and cAMP-induced increase of theintracellular calcium concentration, which in turn triggers a release ofinflammatory mediators (such as histamine) from intracellular granulae,which is a process that is usually referred to as granulocyte“activation.” The concentrations of norketotifen and ketotifen used herehad to be relatively high, since the validated method for in vitrogranulocyte activation uses high concentrations, high temperatures and arelatively long exposure time for the calcium ionophore.

Results

Norketotifen (IC50=9.2 μM) was approximately 10 times more active thanketotifen (IC50=91 μM) in inhibiting A23187-induced granulocyteactivation.

TABLE 11 Inhibition of histamine release (IC50). Test Article IC50 (μM)RS-ketotifen 91 RS-norketotifen 9.2Conclusion

It is concluded that norketotifen was about ten times more potent thanketotifen as an inhibitor of human granulocyte activation, which is ananti-inflammatory effect.

Combinations

Ophthalmic compositions of norketotifen may contain one or moreadditional, therapeutically active ingredients. In addition tonorketotifen, such combination compositions may contain one or moreanti-inflammatory drug, such as for example a steroid belonging to theclass consisting of corticosteroids, such as for example rimexolone(Vexol®, Alcon), fluorometholone (generic), prednisolone acetate(generic), loteprednol etabonate (generic), dexamethasone (generic) anddifluprednate (Durezol™, Sirion), or an NSAID, such as for examplenepafenac (Nevanac™, Alcon), diclofenac (Voltaren™, Novartis), ketorolac(Acular™, Allergan), bromfenac (Xibrom™, Ista), ibuprofen (generic) andindomethacin (generic).

In addition to norketotifen, such combination compositions may containone or more immunosuppressants, such as cyclosporine (generic),tacrolimus (Protopic™, Fujisawa) and pimecrolimus (Elidel®, Novartis).

In addition to norketotifen, such combination compositions may containone or more antimicrobial agents, such as for example aminoglycosides(e.g. neomycin), quinolones (e.g. ofloxacin), macrolides (e.g.azithromycin), polypeptides (e.g. bacitracin), sulfonamides (e.g.sulfacetamide), or combination products (e.g. polymyxin B).

All combination products using compositions described herein areincluded in the present invention.

Equivalents

Norketotifen can also be dissolved and administered in an oil-basecomposition or norketotifen can be dissolved in the oil phase of anoil-in-water emulsion system, which confers certain advantages to thepatient, such as higher drug concentrations, minimal decomposition ofnorketotifen by hydrolysis, and lubrication and improved comfort to theeye.

Norketotifen can also be administered via the nasal route, the oralroute or various parenteral routes using compositions described hereinand devices known to those skilled in the art. Regular or controlledrelease tablets or capsules contain from 1 mg to 20 mg norketotifen.Combinations of routes, such as for example oral doses combined withtopical ocular doses may have advantages, as known to those skilled inthe art of drug formulations.

Micronized norketotifen can be used in ocular compositions ofnorketotifen and have particle sizes where >90 percent of the materialis <10 microns. Also nanoparticles of norketotifen can be used in ocularcompositions thereof and have particle sizes where >90 percent of thematerial is <1 micron.

It may be necessary to adjust the formulations, shown in the examplesherein in order to make said formulations autoclavable. Autoclavableocular formulations are well known and have been described for examplein U.S. Pat. No. 6,776,982 that is hereby incorporated by reference.Autoclavable compositions of norketotifen are included in the presentinvention.

Norketotifen has now been found to be a potent wound healing modulatorand can be used to facilitate ocular surface re-epithelization and toprevent corneal haze resulting from scar formation due to inflammation.Similar effects have previously been described for ketotifen andcyproheptadine (U.S. Pat. No. 5,624,893) and olopatadine (U.S. patentSer. No. 11/947,041). The formulations of norketotifen described hereinare suitable vehicles for the delivery of norketotifen to the eyes ofpatients in need of a wound healing modulator. Norketotifen HF solutionsmay contain concentrations containing about 0.02% to 0.21% norketotifen.Solutions containing solubilizers may have higher concentrations ofnorketotifen. Likewise, emulsions, gels, ointment, suspensions andsimilar formulation may contain concentrations of up to 4%. Allequivalents are included in the present invention.

What is claimed is:
 1. A method of treating a patient suffering fromxerophthalmia, comprising topical administration to the eye of saidpatient in need thereof an ophthalmic formulation, acceptable to theeye, free from any added preservative, and containing a solution ofnorketotifen or a pharmaceutically acceptable salt thereof in aconcentration of 0.001 percent to 0.3 percent by weight, wherein thesolution has a pH of ≧4.5.
 2. The method of claim 1, wherein saidpatient is a member selected from the group consisting of humans, dogsand cats.
 3. The method of claim 1, wherein the formulation exhibitsconstant or declining numbers of cell colonies after up to 4 weeks ofincubation at 25 ° C. with 0.5×10⁵ cells per mL of four out of five ofthe following microorganisms: Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus, Aspergillus niger and Candida albicans.
 4. Themethod of claim 1, wherein the solution is an aqueous solution.
 5. Themethod of claim 1, wherein the ophthalmic formulation is formulated foruse in a multidose container.
 6. The method of claim 1, wherein thesolution has a pH of≧about
 5. 7. The method of claim 6, wherein theophthalmic formulation exhibits constant or declining numbers of cellcolonies after up to 4 weeks of incubation at 24 ° C. with 0.5×10⁵ cellsper mL of Escherichia coli, Pseudomonas aeruginosa, Staphylococcusaureus, Aspergillus niger and Candida albicans.
 8. The method of claim1, wherein the ophthalmic formulation further comprises a chelatingagent, a stabilizing agent, a buffering agent, or a humectant.
 9. Themethod of claim 5, wherein the formulation further comprises EDTA, boricacid and sorbitol.